Reconstitution of Plasmodium Export in Toxoplasma

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DESCRIPTION (provided by applicant): Apicomplexan parasites such as Plasmodium falciparum and Toxoplasma gondii share a common obligate intracellular lifestyle in which the parasite actively penetrates the host cell and resides in a unique membrane-bound vacuole in the cytoplasm of the host. While these parasites are sequestered inside the vacuole, an emerging paradigm is that apicomplexans also deliver proteins into the host cell to modulate the host for optimal intracellular survival. This is particularly important in the intraerythrocytic stges of the Plasmodia, as the red blood cell host is largely metabolically inactive and devoid of many membrane transport systems and organellar functions that can be co-opted in other cells. To compensate for the relative lack of host functions to hijack, Plasmodium efficiently exports hundreds of secretory proteins across the vacuolar membrane and into the host cell that dramatically remodel of the erythrocyte and are key regulators of parasite virulence. Protein export occurs by a specialized vacuolar membrane transport apparatus known as the PTEX translocon, which is unique to Plasmodium and essential for parasite survival. In this proposal, we exploit the similar properties of the secretory pathway and parasitophorous vacuole in apicomplexans to reconstitute the PTEX translocon and export pathway in Toxoplasma. As an initial proof of concept, we have expressed two key components of the translocon, EXP2 and HSP101, in T. gondii and shown that they are secreted to the parasitophorous vacuole membrane. We will build on these results by expressing the remaining three translocon components and assessing their localization and ability to form a complex at the vacuolar membrane. We will also express a Plasmodium host-targeted reporter protein to determine if we can establish a functional PTEX-mediated export pathway in T. gondii. Development of this technology will enable new approaches to dissect the assembly, architecture and function of the PTEX translocon, and also enable the design of novel therapies that specifically target this critical protein export pathway. PUBLIC HEALTH RELEVANCE: Apicomplexan parasites are intracellular parasites that cause important diseases in humans such as malaria (Plasmodium spp) and toxoplasmosis (Toxoplasma gondii). While both of these pathogens hijack their human host cells, Plasmodium has developed a specialized transport system to traffic proteins into the host that is not present in Toxoplasma. While this transport process is difficult to study in Plasmodium because it is essential, we will introduce this pathway into Plasmodium where we will then be able to dissect how this protein superhighway functions.